Many biological materials, such as proteins and whole cells, which are useful for the treatment and prevention of human and animal diseases or as food supplements have a limited shelf life. The limited shelf life is considered to be the result of protein instability at storage temperature. Whilst the shelf life of some proteins and cell cultures may be extended by storing them at low temperature (i.e. 4° C. to 8° C.), shelf lives of less than eighteen months are common.
Biologically active proteins are generally folded in a complex three dimensional manner which is unique to each protein. The proteins are generally organised on three levels; having a primary structure, consisting of a linear chain of covalently bonded amino acid residues (a peptide chain); a secondary structure, in which the peptide chain folds into regular patterns (such as, α helices and β-pleated sheets); and a tertiary structure in which the folded chain further folds upon itself to form a compact structure. In addition, some proteins consist of more than one polypeptide chain held in close arrangement to form what is referred to as the quaternary structure. It is the tertiary and/or the quaternary structure that dictates a protein's ultimate biological activity.
The ultimate structure of a protein may be effected by a number of environmental factors, for example temperature, pH, the presence or absence of certain co-factors or metals, the presence of oxygen, enzymes, oxidising or reducing agents and the presence of water or moisture. Where conditions are not optimal, a protein may not form properly or may denature, such that its biological function is lost, or is at least diminished.
The cells of animals, plants and microorganisms may be considered complex protein materials in the broadest sense as they contain numerous proteins enclosed by a cell membrane and/or cell wall, which membrane or wall inturn presents additional proteins at the cell's surface. As with proteins, the viability of a cell is dependent on the environment in which it resides; for example, temperature, pH, the presence or absence of certain co-factors or metals, presence or absence of certain nutrients, metabolic waste, oxygen, enzymes, oxidising or reducing agents and the presence of water or degree of moisture may individually or collectively act to effect viability.
There are a number of techniques known in the art for stabilising proteins, some of which are briefly discussed below.
Freeze-drying under vacuum (lyophilisation) is commonly used to prepare proteins for use in vaccines and the like. Freeze-drying traditionally involves freezing a solution of the biological protein and removing ice crystals there from by converting them into water vapour under vacuum (sublimation). This process often results in damage to the native structure of the protein.
To help increase the stability of a biological protein being prepared by freeze-drying, additives such as buffering or stabilising agents may be used in the product formulation. However, during freeze-drying, when the temperature of the solution is slowly reduced to minus 20° C. over a period of days, the additives may solidify at different freezing points. As a result, the end product may be a fine puffy cake-like substance actually made up of different layers each representing an individual component. In essence, the additives added to protect the biological protein may be physically and chemically separated there from rendering them useless as protective agents.
An alternative procedure, which is commonly used in the food and dairy industries to make dry fruit concentrates and milk powders, for example, is spray-drying-using-heat. This process involves spraying a fine mist of solution downwards from the top of a spray tower against an upward current of hot air. The hot air removes water from the droplets before they reach the bottom of the tower. Spray drying normally operates at an inlet air temperature exceeding 190° C. and the product temperature may well exceed 60° C. In this operating environment most of the biological protein or cells, such as bacterial cells, denature.
Another protein preparation process known in the art is supercritical fluid drying. In this process, biological agents such as peptides, proteins and nucleic acids are maintained in an aqueous solution until particle formation. The aqueous solvent is removed at the time of particle formation using controlled hydrogen-bonding solvents, such as ethanol, and isopropanol.
Fluid bed spray drying is a modified spray-drying-using-heat technology. The process is commonly used in the pharmaceutical and chemical industry for tablet granulation and/or for drying heat stable materials. The process involves spraying a fine mist of solution containing actives downward from the top of a spray head towards a mass of dry excipients. Simultaneously, an upward current of hot air is passed through the mass of excipients to create a fluidized bed. The hot air removes water from the fluidized wet solids at the bottom of the fluid bed.
Fluid bed spray drying technology may be applicable to pharmaceutical proteins which are heat stable around 50° C. to 60° C. However, the native structure of the protein may be compromised and accordingly the protein may loose all, or at least some, of its biological activity.
Further problems may be associated with fluid bed spray drying as described above; for example, the spray nozzles, which are positioned near the top of the processing chamber, are required to have substantial clearance above the surface of the fluidized bed of excipient materials so that the such materials do not block the spraying nozzles; a substantial amount of the coating material, or liquid containing the active ingredient(s), may block the nozzles' filter system leading to processing loss; and such top spraying fluid bed operation may only be ideal for granulation rather than for spray coating purposes. The fluid bed spray drying process also typically provides coarse and irregular granules having different shapes and sizes. Contact between the granules results in a grinding effect which may result in denaturation of the protein.
Of the techniques in commercial use prior to the development of the present invention, for preparing biological proteins and cells, the technique of microencapsulation may be considered the most useful. Typically, no major equipment is required and the batch size can be as small as 10 g to 20 g thus making it useful for the preparation of biological proteins that may not be plentiful. This process uses organic solvents to solubilize the biological protein which is then encapsulated in polymeric microspheres using either a water-in-oil-in-water (w/o/w) or a solid-in-oil-in-water (s/o/w) emulsion method. Protein is captured into the solid microspheres after water is removed by simple filtration and the solvent is evaporated off.
Microencapsulation technology has been used to make carbon or self-adhesive paper in the paper industry and at least in Japan, food products, such as artificial fish eggs and decorative products are made using gelatine microcapsules to entrap fish or meat flavours.
While microencapsulation may be considered a favourable means to prepare biological proteins and whole cells for storage and future use, the technology is still at the developmental stage in the pharmaceutical and biotechnological industries. The technology has apparent difficulties in that proteins are likely to be denatured by the solvents used and by the necessary emulsifying/homogenising process. In addition, the quality of a product produced according to this process, may be considered undesirable due to the fact that traces of solvent remain in the core of the microcapsules which may hamper the commercialisation of a product produced using this technology.
The present inventor has developed an alternative process based on spray-drying-using heat and microencapsulation technology in which active components, such as proteins, may be stabilised for extended periods.